We have shown that ochratoxin A, which is a secondary metabolite produced by certain strains of Aspergillus ochraceus, is an in vivo inhibitor of renal cortex phosphoenolpyruvate carboxykinase (PEPCK) activity, and gluconeogenesis, in rats. The inhibitor has no effect on the activityof several other proximal tubular enzymes, and is selective towards the kideny in that hepatic PEPCK activity is not affected. This is the first report of an in vivo inhibitor of PEPCK activity. The general goals of the research are two-fold: (a) to determine the mechanism of action of ochratoxin A on PEPCK activity at the protein and mRNA level; and (b) to clone the mRNA from toxin-treated rats that translates in vitro-a 72K protein, and characterize this protein. To accomplish the first objective, I will determine whether the toxin affects PEPCK synthesis or degradation, by using a renal anti-PEPCK antibody. The effect of ochratoxin A on PEPCK mRNA levels will be studied by either cell-free in vitro translation or DNA excess-RNA hybridization on nitrocellulose filters, using a 32P-cDNA complementary to PEPCK mRNA. I will also clone the cDNA for the toxin-induced mRNA by using an enriched poly A(+) mRNA preparation and screen for the particular mRNA by coloby hybridization. This nick-translated 32P-cDNA probe will be used (a) to study the effect of ochratoxin A on the particular mRNA concentration by hybridization onto nitrocellulose; and (b) as a molecular probe of a renal disease in animals and humans, knowns as Balkan endemic nephropathy, that is caused by ochratoxin A. Finally, the ochratoxin A-induced mRNA species will be compared to PEPCK mRNA by hrbrid arrested cell-free translation, using either the 72K protein cDNA or PEPCK cDNA. At the protein level, I will compare the amino acid sequence similarity of the cell-free in vitro induced 72K protein to mitochondrial and cytoplasmic PEPCK, as well as to invitro translated PEPCK, by proteolytic cleavage and peptide mapping.